Inbred corn line CC2

ABSTRACT

An inbred corn line, designated CC2, is disclosed. The invention relates to the seeds of inbred corn line CC2, to the plants and plant parts of inbred corn line CC2 and to methods for producing a corn plant, either inbred or hybrid, by crossing the inbred line CC2 with itself or another corn line. The invention further relates to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other inbred corn lines derived from the inbred CC2.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive corn inbred line,designated CC2. All publications cited in this application are hereinincorporated by reference. There are numerous steps in the developmentof any novel, desirable plant germplasm. Plant breeding begins with theanalysis and definition of problems and weaknesses of the currentgermplasm, the establishment of program goals, and the definition ofspecific breeding objectives. The next step is selection of germplasmthat possess the traits to meet the program goals. The goal is tocombine in a single variety or hybrid an improved combination ofdesirable traits from the parental germplasm. These important traits mayinclude higher yield, resistance to diseases and insects, better stalksand roots, tolerance to drought and heat, reduction of grain moisture atharvest as well as better agronomic quality. With mechanical harvestingof many crops, uniformity of plant characteristics such as germinationand stand establishment, growth rate, maturity and plant and ear heightis important.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

The complexity of inheritance influences choice of breeding method.Backcross breeding is used to transfer one or a few favorable genes fora heritable trait into a desirable cultivar. This approach has been usedextensively for breeding disease-resistant cultivars, nevertheless, itis also suitable for the adjustment and selection of morphologicalcharacters, color characteristics and simply inherited quantitativecharacters such as earliness, plant height or seed size and shape.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s) for three or more years.The best lines are candidates for use as parents in new commercialcultivars; those still deficient in a few traits may be used as parentsto produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a focus on clear objectives.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of corn breeding is to develop new, unique and superior corninbred lines and hybrids. The breeder initially selects and crosses twoor more parental lines, followed by repeated self pollination or selfingand selection, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selfing and mutations.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred lineswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.This unpredictability results in the expenditure of large research fundsto develop a superior new corn inbred line.

The development of commercial corn hybrids requires the development ofhomozygous inbred lines, the crossing of these lines, and the evaluationof the crosses. Pedigree breeding and recurrent selection breedingmethods are used to develop inbred lines from breeding populations.Breeding programs combine desirable traits from two or more inbred linesor various broad-based sources into breeding pools from which inbredlines are developed by selfing and selection of desired phenotypes. Thenew inbreds are crossed with other inbred lines and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable cultivar or inbredline which is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., “Principles of Plant Breeding” John Wiley and Son, pp.115–161, 1960; Allard, 1960; Fehr, 1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F₁progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F₁ hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor exhibited by F₁ hybrids islost in the next generation (F₂). Consequently, seed from hybridvarieties is not used for planting stock.

Hybrid corn seed is typically produced by a male sterility system or byincorporating manual or mechanical detasseling. Alternate strips of twocorn inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent incorn plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile corn and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. These and all patents referred toare incorporated by reference. In addition to these methods, Albertsenet al., U.S. Pat. No. 5,432,068 have developed a system of nuclear malesterility which includes: identifying a gene which is critical to malefertility, silencing this native gene which is critical to malefertility; removing the native promoter from the essential malefertility gene and replacing it with an inducible promoter; insertingthis genetically engineered gene back into the plant; and thus creatinga plant that is male sterile because the inducible promoter is not “on”resulting in the male fertility gene not being transcribed. Fertility isrestored by inducing, or turning “on”, the promoter, which in turnallows the gene that confers male fertility to be transcribed.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran anti-sense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see,Fabinjanski, et al. EPO 89/0301053.8 publication no. 329,308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another version useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, G. R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application, and genotype often limit theusefulness of the approach.

Corn is an important and valuable field crop. Thus, a continuing goal ofplant breeders is to develop stable, high yielding corn hybrids that areagronomically sound. The reasons for this goal are obviously to maximizethe amount of ears or kernels produced on the land used and to supplyfood for both humans and animals. To accomplish this goal, the cornbreeder must select and develop corn plants that have the traits thatresult in superior parental lines for producing hybrids.

SUMMARY OF THE INVENTION

According to the invention, there is provided an inbred corn line,designated CC2. This invention thus relates to the seeds of inbred cornline CC2, to the plants or parts thereof of inbred corn line CC2, toplants or parts thereof having all the physiological and morphologicalcharacteristics of inbred corn line CC2 and to plants or parts thereofhaving all the physiological and morphological characteristics of inbredcorn line CC2 listed in Table 1 and as determined at the 5% significancelevel when grown in the same environmental condition. Parts of theinbred corn plant of the present invention are also provided, such ase.g., pollen obtained from an inbred plant and an ovule of the inbredplant.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of inbred corn plant CC2. The tissue culture willpreferably be capable of regenerating plants having all thephysiological and morphological characteristics of the foregoing inbredcorn plant. Preferably, the cells of such tissue cultures will beembryos, meristematic cells, seeds, callus, pollen, leaves, anthers,roots, root tips, silk, flowers, kernels, ears, cobs, husks, stalks orthe like. Protoplasts produced from such tissue culture are alsoincluded in the present invention. The corn plants regenerated from thetissue cultures are also part of the invention.

Also included in this invention are methods for producing a corn plantproduced by crossing the inbred line CC2 with itself or another cornline. When crossed with itself, i.e. crossed with another inbred lineCC2 plant or self pollinated, the inbred line CC2 will be conserved.When crossed with another, different corn line, an F1 hybrid seed isproduced. F1 hybrid seeds and plants produced by growing said hybridseeds are included in the present invention. A method for producing a F1hybrid corn seed comprising crossing inbred line CC2 corn plant with adifferent corn plant and harvesting the resultant hybrid corn seed arealso part of the invention. The hybrid corn seed produced by the methodcomprising crossing inbred line CC2 corn plant with a different cornplant and harvesting the resultant hybrid corn seed are included in theinvention, as are included the hybrid corn plant or parts thereof, seedsincluded, produced by growing said hybrid corn seed.

In another embodiment, this invention relates to a method for producingthe inbred line CC2 from a collection of seeds, the collectioncontaining both inbred line CC2 seeds and hybrid seeds having CC2 as aparental line. Such a collection of seed might be a commercial bag ofseeds. Said method comprises planting the collection of seeds. Whenplanted, the collection of seeds will produce inbred line CC2 plantsfrom inbred line CC2 seeds and hybrid plant from hybrid seeds. Theplants having all the physiological and morphological characteristics ofcorn inbred line CC2 or having a decreased vigor compared to the otherplants grown from the collection of seeds are identified as inbred lineCC2 parent plants. Said decreased vigor is due to the inbreedingdepression effect and can be identified for example by a less vigorousappearance for vegetative and/or reproductive characteristics includingshorter plant height, small ear size, ear and kernel shape, ear color orother characteristics. As previously mentioned, if the inbred line CC2is self-pollinated, the inbred line CC2 will be preserved, therefore,the next step is controlling pollination of the inbred parent plants ina manner which preserves the homozygosity of said inbred line CC2 parentplant, and the final step is to harvest the resultant seed.

This invention also relates to methods for producing other inbred cornlines derived from inbred corn line CC2 and to the inbred corn linesderived by the use of those methods.

In another aspect, the present invention provides transformed CC2 inbredcorn line or parts thereof that have been transformed so that itsgenetic material contains one or more transgenes, preferably operablylinked to one or more regulatory elements. Also, the invention providesmethods for producing a corn plant containing in its genetic materialone or more transgenes, preferably operably linked to one or moreregulatory elements, by crossing transformed CC2 inbred corn line witheither a second plant of another corn line, or a non-transformed cornplant of the inbred line CC2, so that the genetic material of theprogeny that results from the cross contains the transgene(s),preferably operably linked to one or more regulatory elements. Theinvention also provides methods for producing a corn plant that containsin its genetic material one or more transgene(s), wherein the methodcomprises crossing the inbred corn line CC2 with a second plant ofanother corn line which contains one or more transgene(s) operablylinked to one or more regulatory element(s) so that the genetic materialof the progeny that results from the cross contains the transgene(s)operably linked to one or more regulatory element(s). Transgenic cornplants, or parts thereof produced by the method are in the scope of thepresent invention.

More specifically, the invention comprises methods for producing malesterile corn plants, male fertile corn plants, herbicide resistant cornplants, insect resistant corn plants, disease resistant corn plants,water stress tolerant corn plants, or plants with modified, inparticular decreased, phytate content, plants with modified waxy and/oramylose starch content, plants with modified protein content, plantswith modified oil content or profile, plants with increaseddigestibility or plants with increased nutritional quality. Said methodscomprise transforming the inbred line CC2 corn plant with nucleic acidmolecules that confer male sterility, male fertility, herbicideresistance, insect resistance, disease resistance, water stresstolerance, or that can modify the phytate, the waxy and/or amylosestarches, the protein or the oil contents, the digestibility or thenutritional qualities, respectively. The transformed corn plantsobtained from the provided methods, including male sterile corn plants,male fertile corn plants, herbicide resistant corn plants, insectresistant corn plants, disease resistant corn plants, water stresstolerant corn plants, plants with modified phytate, waxy and/or amylosestarches, protein or oil contents, plants with increased digestibilityand plants with increased nutritional quality are included in thepresent invention. For the present invention and the skilled artisan,disease is understood to be fungal disease, viral disease, bacterialdisease or other plant pathogenic diseases and disease resistant plantwill encompass plants resistant to fungal, viral, bacterial and otherplant pathogens.

Also included in the invention are methods for producing a corn plantcontaining in its genetic material one or more transgenes involved withfatty acid metabolism, carbohydrate metabolism, starch content such aswaxy starch or increased amylose starch. The transgenic corn plantsproduced by these methods are also part of the invention.

In another aspect, the present invention provides for methods ofintroducing one or more desired trait(s) into the corn line CC2 andplants obtained from such methods. The desired trait(s) may be, but notexclusively, a single gene, preferably a dominant but also a recessiveallele. Preferably, the transferred gene or genes will confer suchtraits as male sterility, herbicide resistance, insect resistance,resistance for bacterial, fungal, or viral disease, male fertility,water stress tolerance, enhanced nutritional quality, modified waxycontent, modified amylose content, modified protein content, modifiedoil content, enhanced plant quality, enhanced digestibility andindustrial usage. The gene or genes may be naturally occurring maizegene(s) or transgene(s) introduced through genetic engineeringtechniques. The method for introducing the desired trait(s) ispreferably a backcrossing process making use of a series of backcrossesto the inbred corn line CC2 during which the desired trait(s) ismaintained by selection.

When using a transgene, the trait is generally not incorporated intoeach newly developed line such as CC2 by direct transformation. Rather,the more typical method used by breeders of ordinary skill in the art toincorporate the transgene is to take a line already carrying thetransgene and to use such line as a donor line to transfer the transgeneinto the newly developed line. The same would apply for a naturallyoccurring trait. The backcross breeding process comprises the followingsteps: (a) crossing the inbred line CC2 plants with plants of anotherline that comprise the desired trait(s), (b) selecting the F₁ progenyplants that have the desired trait(s); (c) crossing the selected F₁progeny plants with the inbred line CC2 plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have thedesired trait(s) and physiological and morphological characteristics ofcorn inbred line CC2 to produce selected backcross progeny plants; and(e) repeating steps (c) and (d) one, two, three, four, five six, seven,eight, nine or more times in succession to produce selected, second,third, fourth, fifth, sixth, seventh, eighth, ninth or higher backcrossprogeny plants that comprise the desired trait(s) and all thephysiological and morphological characteristics of corn inbred line CC2as listed in Table 1 and as determined at a 5% significance level whengrown in the same environmental conditions. The corn plants produced bythe methods are also part of the invention. Backcrossing breedingmethods, well known to one skilled in the art of plant breeding will befurther developed in subsequent parts of the specification.

In a preferred embodiment, the present invention provides methods forincreasing and producing inbred line CC2 seed, whether by crossing afirst inbred parent corn plant with a second inbred parent corn plantand harvesting the resultant corn seed, wherein both said first andsecond inbred corn plant are the inbred line CC2 or by planting aninbred corn seed of the inbred corn line CC2, growing an inbred line CC2plant from said seed, controlling a self pollination of the plant wherethe pollen produced by the grown inbred line CC2 plant pollinates theovules produced by the very same inbred line CC2 grown plant andharvesting the resultant seed.

The invention further provides methods for developing corn plants in acorn plant breeding program using plant breeding techniques includingrecurrent selection, backcrossing, pedigree breeding, molecular marker(Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms(RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs). AmplifiedFragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, etc.) enhancedselection, genetic marker enhanced selection and transformation. Cornseeds, plants, and parts thereof produced by such breeding methods arealso part of the invention.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative form of a gene, allof which alleles relates to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing wherein the morphological and physiological characteristicsof an inbred, determined at a 5% significance level when grown in thesame environmental conditions, are recovered in addition to the singlegene transferred into the inbred via the backcrossing technique or viagenetic engineering.

Daily heat unit value. The daily heat unit value is calculated asfollows: (the maximum daily temperature+the minimum daily temperature)/2minus 50. All temperatures are in degrees Fahrenheit. The maximumtemperature threshold is 86 degrees, if temperatures exceed this, 86 isused. The minimum temperature threshold is 50 degrees, if temperaturesgo below this, 50 is used.

HTU. HTU is the summation of the daily heat unit value calculated fromplanting to harvest.

Quantitative Trait Loci (QTL) Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Root Lodging. The root lodging is the percentage of plants that rootlodge; i.e., those that lean from the vertical axis at an approximate30° angle or greater would be counted as root lodged.

Stay Green. Stay green is the measure of plant health near the time ofblack layer formation (physiological maturity). A high score indicatesbetter late-season plant health.

Variety. A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties of plants)

Collection of seeds. In the context of the present invention acollection of seeds will be a grouping of seeds mainly containingsimilar kind of seeds, for example hybrid seeds having the inbred lineof the invention as a parental line, but that may also contain, mixedtogether with this first kind of seeds, a second, different kind ofseeds, of one of the inbred parent lines, for example the inbred line ofthe present invention. A commercial bag of hybrid seeds having theinbred line of the invention as a parental line and containing also theinbred line seeds of the invention would be, for example such acollection of seeds.

Decreased vigor. A plant having a decreased vigor in the presentinvention is a plant that, compared to other plants has a less vigorousappearance for vegetative and/or reproductive characteristics includingshorter plant height, small ear size, ear and kernel shape, ear color orother characteristics.

Inbreeding depression. The inbreeding depression is the loss ofperformance of the inbreds due to the effect of inbreeding, i.e. due tothe mating of relatives or to self-pollination. It increases thehomozygous recessive alleles leading to plants which are weaker andsmaller and having other less desirable traits.

Predicted RM. This trait for a hybrid, predicted relative maturity (RM),is based on the harvest moisture of the grain. The relative maturityrating is based on a known set of checks and utilizes conventionalmaturity such as the Comparative Relative Maturity Rating System or itssimilar, the Minnesota Relative Maturity Rating System.

MN RM. This represents the Minnesota Relative Maturity Rating (MN RM)for the hybrid and is based on the harvest moisture of the grainrelative to a standard set of checks of previously determined MN RMrating. Regression analysis is used to compute this rating.

Yield (Bushels/Acre). The yield is the actual yield of the grain atharvest adjusted to 15.5% moisture.

Moisture. The moisture is the actual percentage moisture of the grain atharvest.

GDU Silk. The GDU silk (=heat unit silk) is the number of growing degreeunits (GDU) or heat units required for an inbred line or hybrid to reachsilk emergence from the time of planting. Growing degree units arecalculated by the Barger Method, where the heat units for a 24-hourperiod are: GDU=((Max Temp+Min Temp)/2)−50 The highest maximum used is86° F. and the lowest minimum used is 50° F. For each hybrid, it takes acertain number of GDUs to reach various stages of plant development.GDUs are a way of measuring plant maturity.

Stalk Lodging. This is the percentage of plants that stalk lodge, i.e.,stalk breakage, as measured by either natural lodging or pushing thestalks and determining the percentage of plants that break off below theear. This is a relative rating of a hybrid to other hybrids forstandability.

Plant Height. This is a measure of the height of the hybrid from theground to the tip of the tassel, and is measured in centimeters.

Ear Height. The ear height is a measure from the ground to the ear nodeattachment, and is measured in centimeters.

Dropped Ears. This is a measure of the number of dropped ears per plot,and represents the percentage of plants that dropped an ear prior toharvest.

Harvest Aspect. This is a visual rating given the day of harvest or theprevious day. Hybrids are rated 1 (poorest) to 9 (best) with poorerscores given for poor plant health, visible signs of fungal infection,poor plant intactness characterized by missing leaves, tassels, or othervegetative parts, or a combination of these traits.

Dry down. This is the rate at which a hybrid will reach acceptableharvest moisture

Pre-anthesis Brittle Snapping. This is a percentage of “snapped” plantsfollowing severe winds prior to anthesis

Pre-anthesis Root Lodging. This is a percentage plants that root lodgeprior to anthesis: plants that lean from the vertical axis at anapproximately 30° angle or greater.

Post-anthesis Root Lodging. This is a percentage plants that root lodgeafter anthesis: plants that lean from the vertical axis at anapproximately 30° angle or greater.

Seedling Vigor. This is the vegetative growth after emergence at theseedling stage, approximately five leaves.

Seed quality. This is a visual rating assigned to the kernels of theinbred. Kernels are rated 1 (poorest) to 9 (best) with poorer scoresgiven for kernels that are very soft and shriveled with splitting of thepericarp visible and better scores for fully formed kernels.

Pollen shed. This is a visual rating assigned at flowering to describethe abundance of pollen produced by the anthers. Inbreds are rated 1(poorest) to 9 (best) with the best scores for inbreds with tassels thatshed more pollen during anthesis.

Plant habit. This is a visual assessment assigned during the latevegetative to early reproductive stages to characterize the plants leafhabit. It ranges from decumbent with leaves growing horizontally fromthe stalk to a very upright leaf habit, with leaves growing nearvertically from the stalk.

Plant intactness. This is a visual assessment assigned to a hybrid orinbred at or close to harvest to indicate the degree that the plant hassuffered disintegration through the growing season. Plants are ratedfrom 1 (poorest) to 9 (best) with poorer scores given for plants thathave more of their leaf blades missing.

Standability. A term referring to the how well a plant remains uprighttowards the end of the growing season. Plants with excessive stalkbreakage and/or root lodging would be considered to have poorstandability.

Late plant greenness. Similar to a stay green rating. This is a visualassessment given at around the dent stage but typically a few weeksbefore harvest to characterize the degree of greenness left in theleaves. Plants are rated from 1 (poorest) to 9 (best) with poorer scoresgiven for plants that have more non-green leaf tissue typically due toearly senescence or from disease.

Silking ability. This is a visual assessment given during flowering.Plants are rated on the amount and timing of silk production. Plants arerated from 1 (poorest) to 9 (best) with poorer scores given for plantsthat produce very little silks that are delayed past pollen shed.

GDU pollen. The number of heat units from planting until 50% of theplants in the hybrid are shedding pollen.

Plant Part. As used herein, the term “plant part” includes leaves,stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs,husks, stalks, root tips, anthers, silk, tissue, cells and the like.

Plant Cell. Plant cell, as used herein includes plant cells whetherisolated, in tissue culture, or incorporated in a plant or plant part.

DETAILED DESCRIPTION OF THE INVENTION

Inbred corn line CC2 is a yellow dent corn inbred with superiorcharacteristics, and provides an excellent female parental line incrosses for producing first generation (F₁) hybrid corn. Inbred cornline CC2 is best adapted to the Central regions of the United StatesCorn Belt commonly referred to as Zones 6, 7 and 8. CC2 can be used toproduce hybrids having a relative maturity of approximately 104 to 113days on the Comparative Relative Maturity Rating System for harvestmoisture of grain. Inbred corn line CC2 shows excellent seedling vigor.CC2 has an abundant yield as a female parent in the production of F₁hybrids. CC2 contributes excellent health to the F₁ hybrids andspecifically grey leaf spot tolerance which results in excellent stalkstrength in the F₁ hybrids along with an appealing look at harvest. Thisin turn allows farmers to harvest more efficiently and reduces fieldlosses compared to hybrids that have inferior stalks and poor planthealth. CC2 also contributes a good yield to moisture ratio to the F₁hybrids when compared to other B73/Stiff Stalk based inbreds. The highyield to moisture ratio and level of production directly correlate to ahigher income per acre for the farmer. CC2 contributes excellent staygreen to the F₁ hybrids which relates directly to higher levels of plantintactness for the farmer at harvest. This plant intactness improvesharvest efficiency.

CC2 is most similar to B73, however, there are numerous differencesincluding the fact that CC2 contributes substantially improved planthealth and stalk characteristics to the F1 hybrid than B73. CC2contributes appreciably higher levels of grey leaf spot tolerance tohybrids than other commonly used stiff stalk derived inbreds such asHC33. Hybrids with CC2 have excellent stay green which correlates to thehigher levels of disease tolerance. CC2 silks emerge with a lower numberof growing degree than is required for the silk emergence for B73.

CC2 is a medium-late season inbred and is very well adapted for use as afemale in seed production. Average heat units to 50% pollen shed areapproximately 1365 and to 50% silk are approximately 1390 as measurednear Champaign, Ill. in 2003 and 2004.

CC2 has a plant height of 250 cm with an average ear insertion of 80 cm.The kernels are arranged in distinct rows on the ear.

CC2 is an inbred line with very high yield potential in hybrids. Hybridswith CC2 as one parental line produce very uniform, large ears. Oftenthese hybrid combinations result in plants which are appreciably betterthan average for overall health, stalk strength and plant intactnesswhen compared to inbred lines of similar maturity and family background.

Some of the criteria used to select ears in various generations include:yield, stalk quality, root quality, disease tolerance with emphasis ongrey leaf spot, test weight, late season plant greenness, late seasonplant intactness, ear retention, ear height, pollen shedding ability,silking ability, and corn borer tolerance. The female seed productioncapabilities were also a factor in the selection of CC2. During thedevelopment of the line, crosses were made to inbred testers for thepurpose of estimating the line's general and specific combining ability,and evaluations were run by the Champaign, Ill. Research Station. Theinbred was evaluated further as a line and in numerous crosses by theChampaign station and other research stations across the Corn Belt. Theinbred has proven to have a good combining ability in hybridcombinations.

The inbred line has shown uniformity and stability for the traits,within the limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in CC2.

Inbred corn line CC2 has the following morphologic and othercharacteristics (based primarily on data collected at Ft. Branch, Ind.and Decatur, Ill.).

TABLE 1 VARIETY DESCRIPTION INFORMATION TYPE: CC2 is a yellow, dent corninbred REGION WHERE DEVELOPED: Champaign, Illinois MATURITY: Days HeatUnits From emergence to 50% of plants in silk: 80 1390 From emergence to50% of plants in pollen: 79 1365 Heat Units: = GDU = ((Max Temp + MinTemp)/2) − 50 PLANT: Plant Height to tassel tip: 250 cm Ear Height tobase of top ear: 80 cm Average Length of Top Ear Internode: 12 cmAverage number of Tillers: 0 Average Number of Ears per Stalk: 1Anthocyanin of Brace Roots: Present, Moderate LEAF: Width of Ear NodeLeaf: 9 cm Length of Ear Node Leaf: 74 cm Number of leaves above topear: 5 Leaf Angle from 2nd Leaf above ear at anthesis to Stalk aboveleaf: 25° Leaf Color: Soft Green Munsell Code 5GY 4/4 Leaf SheathPubescence (Rate on scale from 1 = none to 9 = like peach fuzz): 6Marginal Waves (Rate on scale from 1 = none to 9 = many): 3 LongitudinalCreases (Rate on scale from 1 = none to 9 = many): 3 TASSEL: Number ofLateral Branches: 6 Branch Angle from Central Spike: 25 Degrees TasselLength (from top leaf collar to tassel top): 38 cm Pollen Shed (Rate onscale from 0 = male sterile to 9 = heavy shed): 7 Anther Color: MunsellCode 10RP 6/4 Glume Color: Munsell Code 10RP ⅚ Tassel Glume Bands Color:Absent EAR: (Unhusked Data) Silk Color (3 days after emergence): LightGreen Munsell Code 10Y 9/8 Fresh Husk Color (25 days after 50% silking):Light Green Munsell Code 5GY 8/8 Dry Husk Color (65 days after 50%silking): Munsell Code 5Y 8/8 Position of Ear: Horizontal Husk Tightness(Rate on scale from 1 = very loose to 9 = very tight): 3 Husk Extensionat harvest: Short Husk Extension-Ear Tip Slightly Exposed EAR: (HuskedEar Data) Ear Length: 15.2 cm Ear Diameter at mid-point: 42.3 mm EarWeight: 107.7 gm Number of Kernel Rows: 14–16 Row Alignment: StraightShank Length: 10.0 cm Ear Taper: Slight KERNEL: (Dried) Kernel Length:10.6 mm Kernel Width: 7.6 mm Kernel Thickness: 4.0 mm Round Kernels(Shape Grade): 29% Aleurone Color: White Hard Endosperm Color: Munsellcode 10YR 8/10 Endosperm Type: Dent Weight per 100 kernels (unsizedsample): 23 gm COB: Cob Diameter at Mid-Point: 24 mm Cob Color: PinkAGRONOMIC TRAITS: Stay Green (at 65 days after anthesis) (Rate on scalefrom 1 = worst to 9 = excellent): 5 0% Dropped Ears (at 65 days afteranthesis) 0% Pre-anthesis Brittle Snapping 0% Pre-anthesis Root Lodging0% Post-anthesis Root Lodging (at 65 days after anthesis) Yield ofInbred Per Se (at 12–13% grain moisture): 76 Bu/Acre

FURTHER EMBODIMENTS OF THE INVENTION

This invention is also directed to methods for producing a corn plant bycrossing a first parent corn plant with a second parent corn plantwherein either the first or second parent corn plant is an inbred cornplant of the line CC2. Further, both first and second parent corn plantscan come from the inbred corn line CC2. When self-pollinated, or crossedwith another inbred line CC2 plant, the inbred line CC2 will be stablewhile when crossed with another, different corn line, an F1 hybrid seedis produced.

An inbred line has been produced through several cycles ofself-pollination and is therefore to be considered as a homozygous line.

A hybrid variety is classically created through the fertilization of anovule from an inbred parental line by the pollen of another, differentinbred parental line. Due to the homozygous state of the inbred line,the produced gametes carry a copy of each parental chromosome. As boththe ovule and the pollen bring a copy of the arrangement andorganization of the genes present in the parental lines, the genome ofeach parental line is present in the resulting F₁ hybrid, theoreticallyin the arrangement and organization created by the plant breeder in theoriginal parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross is stable. The F₁ hybrid is then a combination ofphenotypic characteristics issued from two arrangement and organizationof genes, both created by a man skilled in the art through the breedingprocess.

Still further, this invention also is directed to methods for producingan inbred corn line CC2-derived corn plant by crossing inbred corn lineCC2 with a second corn plant and growing the progeny seed, and repeatingthe crossing and growing steps with the inbred corn line CC2-derivedplant from 0 to 7 times. Thus, any such methods using the inbred cornline CC2 are part of this invention: selfing, backcrosses, hybridproduction, crosses to populations, and the like. All plants producedusing inbred corn line CC2 as a parent are within the scope of thisinvention, including plants derived from inbred corn line CC2.Advantageously, the inbred corn line is used in crosses with other,different, corn inbreds to produce first generation (F₁) corn hybridseeds and plants with superior characteristics.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which corn plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, kernels,seeds, ears, cobs, leaves, husks, stalks, roots, root tips, anthers,silk and the like.

Duncan, et al., Planta, 1985, 165:322–332 reflects that 97% of theplants cultured that produced callus were capable of plant regeneration.Subsequent experiments with both inbreds and hybrids produced 91%regenerable callus that produced plants. In a further study in 1988,Songstad, et al., Plant Cell Reports, 1988, 7:262–265, reports severalmedia additions that enhance regenerability of callus of two inbredlines. Other published reports also indicated that “nontraditional”tissues are capable of producing somatic embryogenesis and plantregeneration. K. V. Rao et al., Maize Genetics Cooperation Newsletter,1986, 60:64–65, refers to somatic embryogenesis from glume calluscultures and B. V. Conger, et al., Plant Cell Reports, 1987, 6:345–347indicates somatic embryogenesis from the tissue cultures of corn leafsegments. Thus, it is clear from the literature that the state of theart is such that these methods of obtaining plants are “conventional” inthe sense that they are routinely used and have a very high rate ofsuccess.

Tissue culture of corn is described in European Patent Application,publication 160,390, incorporated herein by reference. Corn tissueculture procedures are also described in Green and Rhodes, Maize forBiological Research—Plant Molecular Biology Association,Charlottesville, Va., 1982, 367–372, and in Duncan et al., Planta,1985,165:322–332. Thus, another aspect of this invention is to providecells which upon growth and differentiation produce corn plants havingthe physiological and morphological characteristics of inbred corn lineCC2.

The utility of inbred corn line CC2 also extends to crosses with otherspecies. Commonly, suitable species will be of the family Graminaceae,and especially of the genera Zea, Tripsacum, Croix, Schlerachne,Polytoca, Chionachne, and Trilobachne, of the tribe Maydeae. Potentiallysuitable for crosses with CC2 may be the various varieties of grainsorghum, Sorghum bicolor (L.) Moench.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed inbred line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedcorn plants, using transformation methods as described below toincorporate transgenes into the genetic material of the corn plant(s).

Expression Vectors for Corn Transformation

Marker Genes—Expression vectors include at least one genetic marker,operably linked to a regulatory element (a promoter, for example) thatallows transformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which, when placed under the control of plant regulatory signals,confers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741–744 (1985), Gordon-Kamm et al., Plant Cell 2:603–618(1990) and Stalker et al., Science 242:419–423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include beta-glucuronidase (GUS), beta-galactosidase,luciferase, and chloramphenicol acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984). Another approach to the identification ofrelatively rare transformation events has been use of a gene thatencodes a dominant constitutive regulator of the Zea mays anthocyaninpigmentation pathway. Ludwig et al., Science 247:449 (1990).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are also available. However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

A gene encoding Green Fluorescent Protein (GFP) has been utilized as amarker for gene expression in prokaryotic and eukaryotic cells. Chalfieet al., Science 263:802 (1994). GFP and mutants of GFP may be used asscreenable markers.

Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain organs,such as leaves, roots, seeds and tissues such as fibers, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as“tissue-preferred”. Promoters which initiate transcription only incertain tissue are referred to as “tissue-specific”. A “cell type”specific promoter primarily drives expression in certain cell types inone or more organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue-specific, tissue-preferred, cell type specific, andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression incorn. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn. With an inducible promoter the rate oftranscription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361–366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90:4567–4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners Gatzet al., Mol. Gen. Genetics 243:32–38 (1994) or Tet repressor from Tn10(Gatz et al., Mol. Gen. Genetics 227:229–237 (1991)). A particularlypreferred inducible promoter is a promoter that responds to an inducingagent to which plants do not normally respond. An exemplary induciblepromoter is the inducible promoter from a steroid hormone gene, thetranscriptional activity of which is induced by a glucocorticosteroidhormone. Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression incorn or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in corn.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810–812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163–171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619–632 (1989) andChristensen et al., Plant Mol. Biol. 18:675–689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581–588 (1991)); MAS (Velten et al., EMBO J.3:2723–2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276–285 (1992) and Atanassova et al., Plant Journal 2 (3):291–300 (1992)).

The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/NcoIfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin corn. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in corn. Plants transformed with a geneof interest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476–482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320–3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723–2729 (1985)and Timko et al., Nature 318:579–582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240–245 (1989)); a pollen-specific promoter such as that from Zm13or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217–224 (1993)). Signal Sequences for TargetingProteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Knox, C., et al.,Plant Mol. Biol. 9:3–17 (1987), Lerner et al., Plant Physiol. 91:124–129(1989), Fontes et al., Plant Cell 3:483–496 (1991), Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell. Biol.108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon, etal., Cell 39:499–509 (1984), Stiefel, et al., Plant Cell 2:785–793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92–6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is corn. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant inbred line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btalpha-endotoxin gene. Moreover, DNA molecules encoding alpha-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus alpha-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Pratt et al., Biochem. Biophys. Res. Comm. 163:1243(1989) (an allostatin is identified in Diploptera puntata). See alsoU.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-beta, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect.

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-alpha-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-alpha-1,4-D-galacturonase. See Lamb et al., BioTechnology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., BioTechnology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT, bar, genes), and pyridinoxy or phenoxy propionicacids and cyclohexones (ACCase inhibitor-encoding genes). See, forexample, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses thenucleotide sequence of a form of EPSP which can confer glyphosateresistance. A DNA molecule encoding a mutant aroA gene can be obtainedunder ATCC accession number 39256, and the nucleotide sequence of themutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Europeanpatent application No. 0 333 033 to Kumada et al., and U.S. Pat. No.4,975,374 to Goodman et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a PAT gene is provided inEuropean application No. 0 242 246 to Leemans et al. DeGreef et al.,BioTechnology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for PAT activity. Exemplary ofgenes conferring resistance to phenoxy propionic acids and cyclohexones,such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3genes described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knutzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992)

B. Increased resistance to high light stress such as photo-oxidativedamages, for example by transforming a plant with a gene coding for aprotein of the Early Light Induced Protein family (ELIP) as described inWO 03074713 in the name of Biogemma.

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., BioTechnology 10:292 (1992) (production of transgenic plantsthat express Bacillus licheniformis alpha-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley alpha-amylase gene), and Fisher et al., PlantPhysiol. 102:1045 (1993) (maize endosperm starch branching enzyme II).

D. Increased resistance/tolerance to water stress or drought, forexample, by transforming a plant to create a plant having a modifiedcontent in ABA-Water-Stress-Ripening-Induced proteins (ARS proteins) asdescribed in WO 0183753 in the name of Biogemma, or by transforming aplant with a nucleotide sequence coding for a phosphoenolpyruvatecarboxylase as shown in WO02081714. The tolerance of corn to drought canalso be increased by an overexpression of phosphoenolpyruvatecarboxylase (PEPC-C4), obtained, for example from sorghum.

E. Increased content of cysteine and glutathione, useful in theregulation of sulfur compounds and plant resistance against variousstresses such as drought, heat or cold, by transforming a plant with agene coding for an Adenosine 5′ Phosphosulfate as shown in WO 0149855.

F. Increased nutritional quality, for example, by introducing a zeingene which genetic sequence has been modified so that its proteinsequence has an increase in lysine and proline. The increasednutritional quality can also be attained by introducing into the maizeplant an albumin 2S gene from sunflower that has been modified by theaddition of the KDEL peptide sequence to keep and accumulate the albuminprotein in the endoplasmic reticulum.

G. Decreased phytate content: 1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize, this, for example, could beaccomplished, by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

Methods for Corn Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67–88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89–119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and corn. Hiei etal., The Plant Journal 6:271–282 (1994) and U.S. Pat. No. 5,591,616issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 microm. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299(1988), Klein et al., Biotechnology 6:559–563 (1988), Sanford, J. C.,Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., BioTechnology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. D'Halluin et al., Plant Cell 4:1495–1505 (1992) andSpencer et al., Plant Mol. Biol. 24:51–61 (1994).

Following transformation of corn target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic inbred line. The transgenic inbred line couldthen be crossed, with another (non-transformed or transformed) inbredline, in order to produce a new transgenic inbred line. Alternatively, agenetic trait which has been engineered into a particular corn lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-eliteinbred line into an elite inbred line, or from an inbred line containinga foreign gene in its genome into an inbred line or lines which do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross, or the process of backcrossing, depending on the context.

When the term inbred corn plant is used in the context of the presentinvention, this also includes any inbred corn plant where one or moredesired trait has been introduced through backcrossing methods, whethersuch trait is a naturally occurring one or a transgenic one.Backcrossing methods can be used with the present invention to improveor introduce one or more characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the gene or the genes for thedesired characteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Fehr, 1987).

In a typical backcross protocol, the original inbred of interest(recurrent parent) is crossed to a second inbred (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a corn plant isobtained wherein all the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, generally determined at a 5% significance level when grown in thesame environmental conditions, in addition to the gene or genestransferred from the nonrecurrent parent. It should be noted that some,one, two, three or more, self-pollination and growing of a populationmight be included between two successive backcrosses. Indeed, anappropriate selection in the population produced by theself-pollination, i.e. selection for the desired trait and physiologicaland morphological characteristics of the recurrent parent might beequivalent to one, two or even three additional backcrosses in acontinuous series without rigorous selection, saving time, money andeffort for the breeder. A non limiting example of such a protocol wouldbe the following: a) the first generation F₁ produced by the cross ofthe recurrent parent A by the donor parent B is backcrossed to parent A,b) selection is practiced for the plants having the desired trait ofparent B, c) selected plants are self-pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and the physiological and morphologicalcharacteristics of parent A, d) the selected plants are backcrossed one,two, three, four, five or more times to parent A to produce selectedbackcross progeny plants comprising the desired trait of parent B andthe physiological and morphological characteristics of parent A. Step c)may or may not be repeated and included between the backcrosses of stepd.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass not only visualinspection and simple crossing, but also follow up of thecharacteristic(s) through genetically associated markers and molecularassisted breeding tools. For example, selection of progeny containingthe transferred trait is done by direct selection, visual inspection fora trait associated with a dominant allele, while the selection ofprogeny for a trait that is transferred via a recessive allele, such asthe waxy starch characteristic, require selfing the progeny to determinewhich plant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, i.e. they may be naturally present in the non recurrentparent, examples of these traits include but are not limited to, malesterility, waxy starch, amylose starch, herbicide resistance, resistancefor bacterial, fungal, or viral disease, insect resistance, malefertility, water stress tolerance, enhanced nutritional quality,industrial usage, increased digestibility yield stability and yieldenhancement. An example of this is the Rp1D gene which controlsresistance to rust fungus by preventing P. sorghi from producing spores.The Rp1D gene is usually preferred over the other Rp genes because it iswidely effective against all races of rust, but the emergence of newraces has lead to the use of other Rp genes comprising, for example, theRp1E, Rp1G, Rp1I, Rp1K or “compound” genes which combine two or more Rpgenes including Rp1GI, Rp1GDJ, etc. These genes are generally inheritedthrough the nucleus. Some known exceptions to this are the genes formale sterility, some of which are inherited cytoplasmically, but stillact as single gene traits. Several of these single gene traits aredescribed in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.

In 1981, the backcross method of breeding accounted for 17% of the totalbreeding effort for inbred line development in the United States,according to, Hallauer, A. R. et al. (1988) “Corn Breeding” Corn andCorn Improvement, No. 18, pp. 463–481. The backcross breeding methodprovides a precise way of improving varieties that excel in a largenumber of attributes but are deficient in a few characteristics (Allard(1960), Principles of Plant Breeding, John Wiley & Sons, Inc.). Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing, the gene or genes being transferred unlike all other geneswill be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a variety with exactly the adaptation, yielding ability andquality characteristics of the recurrent parent but superior to thatparent in the particular characteristic(s) for which the improvementprogram was undertaken. Therefore, this method provides the plantbreeder with a high degree of genetic control of his work.

The method is scientifically exact because the morphological andagricultural features of the improved variety could be described inadvance and because the same variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method”, 1930 Jour. Amer. Soc.Agron., 22: 289–244).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will bemodified only with regards to genes being transferred, which aremaintained in the population by selection.

Examples of successful backcrosses are the transfer of stem rustresistance from “Hope” wheat to “Bart” wheat and the transfer of buntresistance to “Bart” wheat to create “Bart 38” which has resistance toboth stem rust and bunt. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in “CaliforniaCommon” alfalfa to create “Caliverde”. This new “Caliverde” varietyproduced through the backcross process is indistinguishable from“California Common” except for its resistance to the three nameddiseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,colour characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, “Calady”, has been produced by Jones andDavis. In dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e. grain size. “Lady Wright”, along grain variety was used as the donor parent and “Coloro”, a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety “Calady” was produced.

INDUSTRIAL APPLICABILITY

Corn is used as human food, livestock feed, and as raw material inindustry. The food uses of corn, in addition to human consumption ofcorn kernels, include both products of dry- and wet-milling industries.The principal products of corn dry-milling are grits, meal and flour.The corn wet-milling industry can provide corn starch, corn syrups, anddextrose for food use. Corn oil is recovered from corn germ, which is aby-product of both dry- and wet-milling industries.

Corn, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs and poultry. Industrial uses of corn include production ofethanol, corn starch in the wet-milling industry and corn flour in thedry-milling industry. The industrial applications of corn starch andflour are based on functional properties, such as viscosity, filmformation, adhesive properties, and ability to suspend particles. Cornstarch and flour have application in the paper and textile industries.Other industrial uses include applications in adhesives, buildingmaterials, foundry binders, laundry starches, explosives, oil-well mudsand other mining applications.

Plant parts other than the grain of corn are also used in industry, forexample, stalks and husks are made into paper and wallboard and cobs areused for fuel and to make charcoal.

The seed of inbred corn line CC2, the plant produced from the inbredseed, the hybrid corn plant produced from the crossing of the inbred,hybrid seed, and various parts of the hybrid corn plant and transgenicversions of the foregoing, can be utilized for human food, livestockfeed, and as a raw material in industry.

TABLES

In the tables that follow, the traits and characteristics of hybridcombination having inbred corn line CC2 as a parental line are givencompared to other hybrids. The data collected are presented for keycharacteristics and traits. The field tests have been made at numerouslocations, with one or two replications per location. Information aboutthese hybrids as compared to the check hybrids is presented.

The first pedigree listed in the comparison group is the hybrid(s)containing CC2. MON810 is the designation given by the Monsanto Company(St. Louis, Mo.) for the transgenic event that, when expressed in maize,produces an endotoxin that is efficacious against the European cornborer, Ostrinia nubilalis, and certain other Lepidopteran larvae.Information for each pedigree includes:

1. Mean yield of the hybrid across all locations (bu/ac) is show incolumn 2.

2. A mean for the percentage moisture (H2O Grain) for the hybrid acrossall locations is shown in column 3.

3. A mean of the yield divided by the percentage moisture (Y/M) for thehybrid across all locations is shown in column 4.

4. A mean of the percentage of plants with stalk lodging (SL %) acrossall locations is shown in column 5.

5. A mean of the percentage of plants with root lodging (RL %) acrossall locations is shown in column 6.

6. Test weight is the grain density as measured in pounds per bushel andis shown in column 7.

7. Stay Green is a rating made by a trained person as the hybridsapproach maturity. A scale of 1=Lowest to 9=Highest/Most Desirable isused and is listed in column 8.

8. Harvest Appearance is a rating made by a trained person on the dateof harvest. Harvest appearance is the rater's impression of the hybridbased on, but not limited to, a combination of factors to include plantintactness, tissue health appearance and ease of harvest as it relatesto stalk lodging and root lodging. A scale of 1=Lowest to 9=Highest/MostDesirable is used and is listed in column 9

9. Population is the actual counted plants per harvested plot areacalculated on a plants per acre basis and is shown in column 10.

10. Plant Height is a physical measurement taken from the ground levelto the tip of the tassel. It is expressed to the nearest inch and isshown in column 11.

11. Ear Height is a physical measurement taken from the ground level tothe node of attachment for the upper ear. It is expressed to the nearestinch and is shown in column 12.

TABLE 2 Overall Comparisons: 2004/31 Locations Yld H2O TW Har PlHt EHtPedigree Grain Grain Y/M SL % RL % lbs. SG Ap Pop Inch InchCC2xLH287BtMon810 207.3 19.6 11.2 1.4 8.1 55.6 6.7 6.0 28788 124 43P33P67 202.4 20.6 10.3 1.6 4.7 57.4 6.5 6.2 27781 123 53 P33B51 201.820.0 10.5 3.1 4.7 56.9 5.6 6.1 27716 118 48 LH331xLH283BtMon810 193.719.0 10.7 0.8 6.8 57.4 7.2 6.7 27869 116 49 HC33xLH287+863 193.4 17.911.2 2.5 9.9 57.3 5.5 5.1 28569 120 41 HC33RRxLH287BtMon810 191.2 18.011.0 2.8 9.9 57.0 5.6 5.4 28538 124 41 HC33ALS2xLH287BtMon810 189.1 18.610.6 2.2 8.7 56.8 5.5 5.6 28370 117 38 HC33RRxLH283BtMon810 182.8 19.49.9 2.4 11.3 57.5 6.8 6.2 27762 112 44 LH332xLH283BtMon810 180.7 19.19.9 2.1 13.2 57.4 6.8 5.8 27700 114 47 SGI890HXxLH287 205.7 20.7 10.41.8 6.2 55.8 6.4 6.0 28021 119 46 TR7245xSGI847HX 199.3 19.5 10.7 1.62.6 56.3 6.9 6.0 27477 118 50

TABLE 3 Overall Comparisons: 2004/32 Locations Yld H2O TW Har PlHt EHtPedigree Grain Grain Y/M SL % RL % lbs. SG Ap Pop Inch InchCC2xLH287BtMon810 205.9 19.3 11.3 2.8 8.0 55.2 6.4 6.1 27669 112 39P33P67 193.6 20.3 10.0 2.9 2.1 57.6 6.3 6.3 27746 109 46 LH332xLH324200.4 18.7 11.1 2.7 6.7 56.5 6.9 6.0 27485 103 45

TABLE 4 Overall Comparisons: 2003/27 Locations Yld H2O TW Har PlHt EHtPedigree Grain Grain Y/M SL % RL % lbs. SG Ap Pop Inch InchCC2xLH287BtMon810 186.4 20.4 9.3 3.5 8.3 52.2 6.4 6.4 29289 117 42HC33xLH287BtMon810 175.7 19.8 9.1 8.0 10.1  53.4 5.5 5.9 29528 116 42P33P67 184.4 22.7 8.2 4.4 2.6 55.5 6.0 6.1 29297 117 50 P31G98 172.222.3 8.1 4.4 5.7 54.0 6.7 5.1 29689 118 50 HC33xLH283 169.1 20.4 8.6 3.22.1 55.0 6.3 5.5 29161 110 42 FR3318xLH283 176.5 22.5 8.2 5.7 4.4 54.76.6 5.4 29607 110 45 LH311xLH321 164.8 21.8 7.8 2.0 0.0 53.9 5.2 4.829195 101 38

Table 5 below lists the ratings for grey leaf spot resistance. Theratings were conducted by Professional Seed Research, Inc. located at7S437 Dugan Road, Sugar Grove, Ill. 60554. Professional Seed Research isrecognized in the seed industry as a reliable source for pathogenicevaluation of corn inbreds and hybrids. In the table below, CC2 and HC33are compared in hybrid combination using a common tester line, LH287Bt,as the other parent in the F1 hybrid. The data represent ninereplications. Definitions for Table 5:

-   1=Highly resistant, no or very limited symptoms despite exposure to    pathogen-   2=Moderately resistant, lesions limited in number and or size, no    significant damage-   3=Intermediate resistance, several lesions, expect some damage in    epidemic-   4=Susceptible, multiple lesions of greater size, sufficient to    reduce yield and stalk quality when Midwest disease pressure is high-   5=Very susceptible, considerable damage expected when Midwest    disease pressure is high.

TABLE 5 Hybrid GLS GLS CC2xLH287BtMon810 2 2 HC33xLH287BtMon810 4 4

DEPOSIT INFORMATION

A deposit of the KWS Kleinwanzlebener Saatzucgt AG and LimagrainVerneuil Holding SA proprietary inbred corn line CC2 disclosed above andrecited in the appended claims has been made with the NationalCollections of Industrial Food and Marine Bacteria (NCIMB), FergusonBuilding, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, Scotland,United Kingdom. The date of deposit was Feb. 20, 2006. The deposit of2,500 seeds was taken from the same deposit maintained by AgReliantGenetics since prior to the filing date of this application. Allrestrictions upon the deposit have been removed, and the deposit isintended to meet all of the requirements of 37 C.F.R. §1.801–1.809. TheNCIMB accession number is NCIMB 41376. The deposit will be maintained inthe depository for a period 30 years, or 5 years after the last request,or for the effective life of the patent, whichever is longer, and willbe replaced as necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationssuch as single gene modifications and mutations, somaclonal variants,variant individuals selected from large populations of the plants of theinstant inbred and the like may be practiced within the scope of theinvention, as limited only by the scope of the appended claims.

1. A seed of corn inbred line designated CC2, a representative sample ofseed of said line having been deposited under NCIMB No.
 41376. 2. A cornplant, or a part thereof, produced by growing the seed of claim
 1. 3. Acorn plant, or a part thereof, having all the physiological andmorphological characteristics of the inbred line CC2, a representativesample of seed of said line having been deposited under NCIMB No. 41376.4. A tissue culture of regenerable cells produced from the plant ofclaim 2, wherein cells of the tissue culture are from a plant partselected from the group consisting of leaves, pollen, embryos, roots,root tips, anthers, silks, flowers, kernels, ears, cobs, husks, seedsand stalks.
 5. A corn plant regenerated from the tissue culture of claim4, said plant having all the morphological and physiologicalcharacteristics of inbred line CC2, representative seed of said linehaving been deposited under NCIMB No.
 41376. 6. A method for producing ahybrid corn seed wherein the method comprises crossing the plant ofclaim 2 with a different corn plant and harvesting the resultant hybridcorn seed.
 7. A method for producing inbred line CC2, a representativesample of seed of said line having been deposited under NCIMB No. 41376,wherein the method comprises: a) planting a collection of seedscomprising seed of a hybrid, one of whose parents is inbred line CC2,said collection also comprising seed of said inbred; b) growing plantsfrom said collection of seeds; c) identifying the plants having thephysiological and morphological characteristics of corn inbred line CC2as inbred parent plants; d) controlling pollination of said inbredparent plants in a manner which preserves the homozygosity of saidinbred parent plant; and e) harvesting the resultant seed.
 8. The methodof claim 7 wherein step (c) comprises identifying plants with decreasedvigor compared to the other plants grown from the collection of seeds.9. A method for producing a corn plant that contains in its geneticmaterial one or more transgenes, which when expressed, confers a traitselected from the group consisting of male sterility, male fertility,herbicide resistance, insect resistance, disease resistance, waterstress tolerance and increased digestibility, wherein the methodcomprises crossing the corn plant of claim 2 with either a second plantof another corn line which contains the transgene(s) or a transformedcorn plant of the inbred corn line CC2 which contains the transgene(s),so that the genetic material of the progeny that results from the crosscontains the transgene(s) operably linked to a regulatory element.
 10. Acorn plant, or a part thereof, produced by the method of claim
 9. 11.The corn plant of claim 10, wherein the transgene confers resistance toan herbicide selected from the group consisting of imidazolinone,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile.
 12. The corn plant of claim 10, wherein the transgeneencodes a Bacillus thuringiensis protein.
 13. A method of producing acorn plant with waxy starch or increased amylose starch wherein themethod comprises transforming the corn plant of claim 2 with a transgenethat modifies the carbohydrate metabolism.
 14. A corn plant produced bythe method of claim
 13. 15. A method of introducing a desired trait intocorn inbred line CC2 wherein the method comprises: (a) crossing theinbred line CC2 plants grown from the inbred line CC2 seed, arepresentative seed sample of seed of said line having been depositedunder NCIMB No. 41376, with plants of another corn line that comprise adesired trait to produce F₁ progeny plants, wherein the desired trait isselected from the group consisting of male sterility, male fertility,herbicide resistance, insect resistance, disease resistance, waxystarch, water stress tolerance, increased amylose starch and increaseddigestibility; (b) selecting F₁ progeny plants that have the desiredtrait to produce selected F1 progeny plants; (c) crossing the selectedF₁ progeny plants with the inbred line CC2 plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have thedesired trait and physiological and morphological characteristics ofcorn inbred line CC2 listed in Table 1 to produce selected backcrossprogeny plants; and (e) repeating steps (c) and (d) three or more timesin succession to produce selected fourth or higher backcross progenyplants that comprise the desired trait and the physiological andmorphological characteristics of corn inbred line CC2 as listed in Table1 and as determined at a 5% significance level when grown in the sameenvironmental conditions.
 16. A corn plant produced by the method ofclaim 15, wherein the plant has the desired trait and all of thephysiological and morphological characteristics of corn inbred line CC2as listed in Table 1 and as determined at a 5% significance level whengrown in the same environmental conditions.
 17. A method for producinginbred line CC2 seed, a representative sample of seed of said linehaving been deposited under NCIMB No. 41376, wherein the methodcomprises crossing a first inbred parent corn plant with a second inbredparent corn plant and harvesting the resultant corn seed, wherein bothsaid first and second inbred corn plant are the corn plant of claim 3.18. A method for producing inbred line CC2 seed, a representative sampleof seed of said line having been deposited under NCIMB No. 41376,wherein the method comprises: a) planting an inbred corn seed of claim1; b) growing plant from said seed; c) controlling pollination in amanner that the pollen produced by the grown plant pollinates the ovulesproduced by the grown plant; and d) harvesting the resultant seed.